Biomedical subjects

M L Mello

Publications and source records attributed to M L Mello.

At least 19 recordsLinked to original sources

PubMed2004

Experimentally induced heat- and cold-shock tolerance in adult Panstrongylus megistus (Burmeister) (Hemiptera, Reduviidae).

The survival rate of domestic male and female adult Panstrongylus megistus was studied after sequential heat and cold shocks in order to investigate shock tolerance compared to that previously reported for nymphs. Sequential shocks were such that a milder shock (0 degree C, 5 degrees C, 35 degrees C, or 40 degrees C for 1 h) preceded a severe one (0 degree C or 40 degrees C for 12 h), separated by intervals of 8, 18, 24, and 72 h at 28 degrees C (control temperature). The preliminary thermal shock induced tolerance to the more severe one, although tolerance intensity depended on the initial shock temperature and the interval between treatments. Despite the observed tolerance, the survival rate for insects subjected to both shocks decreased when compared to that of individuals subjected to a single mild shock. When tolerance differed with sex, females showed greater values than males. In contrast to the response detected in nymphs, for which higher heat tolerance values were sustained for intervals of up to 24 h (preliminary shock, 35 degrees C) or even longer (preliminary shock, 40 degrees C) between sequential shocks, significant values were verified in adults only for shock intervals of up to 8 h (preliminary shock, 40 degrees C). While findings for nymphs exhibited considerable cold-shock tolerance under conditions in which preliminary shocks were given at 5 degrees C or 0 degree C and the periods between shocks were up to 72 h long, the adults were shown to be capable of acquiring a substantial tolerance response to a more severe cold shock only when the preliminary shock was given at 0 degree C and shock interval surpassed 18 h. It is assumed that the mechanisms involved in the cellular protection of P. megistus under sequential temperature shocks (heat shock protein action?) may loose effectiveness with insect development.

Animals↗

PubMed2001

Effect of sequential cold shocks on survival and molting incidence in Panstrongylus megistus (Burmeister) (Hemiptera, Reduviidae).

The survival and molting incidence were studied in the insect, Panstrongylus megistus, following sequential cold shocks in which a milder shock at 0 or 5 degrees C for 1 h preceded a more severe shock (0 degrees C, 12 h). The shocks were separated by intervals of 8, 18, 24, and 72 h at 28 degrees C. The survival rate after sequential shocks was identical to that of unshocked controls. Cold-shock tolerance differed from heat-shock tolerance since the latter varied with the time between shocks and was much more transient. Sequential cold shocks produced a higher molting incidence when the first shock was given at 0 compared to 5 degrees C. This response was more rapid than that to sequential heat shocks. Cold-shock tolerance in P. megistus may involve heat-shock proteins, although other protective mechanisms may also occur concurrently. This is the first report of cold-shock tolerance in a blood-sucking hemipteran.

Animals↗

PubMed2001

Cell death and survival alterations in Malpighian tubules of Triatoma infestans following heat shock.

In this study, we examined cell survival and cell death in response to heat shock in an insect organ composed of highly polyploid cells no longer capable of cell division. For this, the frequency of nuclear phenotypes in Feulgen-stained Malpighian tubules of the blood-sucking insect, Triatoma infestans, was analyzed at various times after a short heat shock with or without subsequent moderate fasting. Cell death DNA fragmentation was studied immunocytochemically. Normal phenotypes and phenotypes indicative of cell survival (heterochromatin decondensation, nuclear fusion) and death (apoptosis, necrosis) were observed, especially in heat-shocked specimens. While the number of total and normal nuclei decreased following heat shock, the frequency of apoptosis increased during a short period (7 days) after heat shock. During a 30-day period following heat shock, the frequency of necrosis in fasted but not in fully nourished nymphs increased simultaneously with a decrease in the frequency of apoptosis. This finding suggests that the stress promoted by heat shock, but not that associated with heat shock plus fasting, can be dealt with by the apoptosis program. When considering the forms of cell survival, heterochromatin decondensation was more relevant in fully nourished nymphs, whereas nuclear and cell fusions were more important in fasted specimens. The forms of cell survival and cell death reported here may have protected the organ from damage by the stressing agents. In cells with no induction or accumulation of heat-shock proteins, cell death and the forms of cell survival observed here were the probable consequence.

Animals↗

PubMed2001

RNA relocation and persistence of nucleolus-like bodies at mitosis in benzo[a]pyrene-transformed human breast epithelial cells after microcell-mediated transfer of chromosomes 11 and 17.

RNA relocation and the incidence of nucleolus-like bodies accumulated during mitosis were studied cytochemically in benzo[a]pyrene (BP)-transformed human breast epithelial MCF-10F cells after microcell-mediated transfer of normal chromosomes 11 and 17. The changes resulting from the transfer of these two chromosomes in tumorigenic MCF-10F cells (BP1-E cell line) were examined, since alterations in these chromosomes are involved in the expression of the transformed and tumorigenic phenotypes in the MCF-10F cell series. In addition, the frequency of nucleolus-like bodies decreases drastically with transformation and tumorigenicity in MCF-10F cells, thus being conceivable that it would be affected in presence of normal chromosomes 11 or 17. The pattern of RNA relocation associated with the mitotic spindle did not vary in the cell lines analyzed. The introduction of chromosome 17 in BP1-E cells either decreased or did not affect the frequency of persistent nucleolus-like bodies. In contrast, in cells which received a normal chromosome 11, the frequency of nucleolus-like bodies was closer to that of non-transformed MCF-10F cells. These results suggest that a normal chromosome 11 but not chromosome 17 contributes to the maintenance of an RNA surplus which accumulates in nucleolus-like bodies during cell division of the human breast epithelial cells, at least in vitro. Some loci which were retained in the BP1-E cells which received a normal chromosome 11 are probably involved with the control of RNA transcript production. Figure 1 on http://www.esacp.org/acp/2001/23-3,4/mello.htm

Benzo(a)pyrene↗

PubMed2001

Experimentally induced heat-shock tolerance in Panstrongylus megistus (Hemiptera: Reduviidae).

The survival and molting incidence of fifth-instar nymphs of Panstrongylus megistus (Burmeister, 1835), a vector of Chagas' disease, were investigated following sequential heat shocks in which a mild shock (35 or 40 degrees C, 1 h) preceded a more drastic one (40 degrees C, 12 h). The shocks were separated by 8-, 18-, 24-, or 72-h periods at 28 degrees C. The heat-shock tolerance response was more effective when the first shock was given at 40 degrees C. When the period between shocks was 18 h, the tolerance to sequential shocks (in terms of specimen survival) weakened, which suggested a transient control of the process that enables the organism to circumvent the unfavorable effects of severe shock. In terms of molting incidence, the heat-shock tolerance was only demonstrated when the period between the first shock at 40 degrees C for 1 h and the second shock at 40 degrees C for 12 h was > or = 24 h. These results are the first to demonstrate the induction of heat-shock tolerance in a blood-sucking hemipteran.

Animals↗

PubMed2000

Restriction enzyme analysis of DNA methylation in "condensed" chromatin of Ha-ras-transformed NIH 3T3 cells.

Increased amounts of chromatin condensation (i.e., localized areas of high DNA density, or chromatin higher order packing state) have been described in NIH 3T3 cells transformed with the Ha-ras oncogene. The structural basis for this oncogene-mediated alteration in nuclear organization is unknown. Since DNA methylation is likely to be involved in regulating the nucleosomal level of DNA packaging, we studied the role of DNA methylation in higher-order chromatin organization induced by Ha-ras. CpG-methylated DNA content was estimated in "condensed" chromatin of Ha-ras-transformed NIH 3T3 cell lines which differ in ras expression and ras-induced metastatic ability but present approximately the same values of "condensed" chromatin areas. The question posed was that if DNA methylation were involved with the chromatin higher-order organization induced by Ha-ras in these cell lines, the methylated DNA density in the "condensed" chromatin would also be the same. The DNA evaluation was performed by video image analysis in Feulgen-stained cells previously subjected to treatment with Msp I and Hpa II restriction enzymes, which distinguish between methylated and non-methylated DNA. The amount of methylated CpG sequences not digested by Hpa II in "condensed" chromatin regions was found to vary in the studied ras-transformed cell lines. DNA CpG methylation status is thus suggested not to be involved with the higher order chromatin condensation induced by ras transformation in the mentioned NIH 3T3 cell lines.

3T3 Cells↗

PubMed1999

Apoptosis and catastrophic cell death in benzo[a]pyrene-transformed human breast epithelial cells.

Apoptosis and mitotic death, bi- and multinucleation, giant cells and micronucleation were investigated in human breast epithelial cell lines transformed by benzo[a]pyrene (BP) (BP1, BP1-E and BP1-E1 cells) and in BP1 cells transfected with the c-Ha-ras oncogene (BP1-Tras cells). Since BP induces apoptosis and the abnormal expression of ras genes elicits catastrophic mitosis, both cell death phenomena were expected to occur in this system, especially in BP1-Tras cells. Regardless of the cell line considered, single-nucleate cells were found to be eliminated preferentially through apoptosis, while bi- and multinucleate cells were eliminated through catastrophic mitosis. Apoptosis and catastrophic mitosis were observed in all cell lines but were significantly more frequent in BP1-Tras cells. The abnormal expression of Ha-ras in the latter cells may enhance in this system the effects of the BP apoptosis path reported for BP-transformed Hepa 1c1c7 hepatoma cells. Transfection with the ras oncogene also enhanced the mitotic disturbances, which produced multi- and micronucleation and mitotic death, possibly because of the genomic instability promoted by this oncogene in the BP-transformed cell line.

Apoptosis↗

PubMed1999

RNA relocation at mitosis in transformed and tumorigenic human breast epithelial cells.

The fate of RNA revealed by metachromatic staining after a critical electrolyte concentration assay using toluidine blue and Mg(2+)ions as competitors for the substrate dye binding sites was followed at mitosis in human breast epithelial cells transformed by benzo[a]pyrene and transfected with the c-Ha- ras oncogene. The aim was to detect changes in RNA distribution during mitosis in human transformed/tumorigenic cells exhibiting increased nucleolar sizes and rRNA production while in interphase. RNA relocation in association with the mitotic spindle fibers was observed from metaphase to telophase not to vary in all the cell lines studied. RNA-containing nucleolus-like bodies persistent during mitosis were found to decrease in frequency in the transformed and tumorigenic cells in comparison with control non-transformed cells simultaneously to the previously reported increase in nucleolar areas for the same cells while in interphase. It is suggested that an improved use of RNA transcripts has been developed with cell transformation and tumorigenesis in this particular model.

Benzo(a)pyrene↗

PubMed1999

Ha-ras oncogene effect on DNA content and chromatin supraorganization in benzo[a]pyrene-transformed human breast epithelial cells.

When transfected to benzo[a]pyrene (BP)-transformed MCF-10F human breast epithelial cells (BP1 cell line) the c-Ha-ras oncogene has proven to enhance the neoplastic changes initiated by exposure to BP, giving rise to an aggressive tumorigenic cell line, BP1-Tras. We have previously demonstrated by image analysis that BP affects the DNA content and the chromatin supraorganization of MCF-10F cells. Here Feulgen-stained BP1-Tras cells were studied by image analysis in order to evaluate possible additional changes in DNA content and chromatin texture induced by insertion of the ras oncogene. A high variability in DNA content also including polyploidy or near-polyploidy, and an increase in the packing states of the chromatin which became still condensed in BP1 cells were found in BP1-Tras cells. The results differed from those reported for the BP1-E1 cell line which is also an aggressive tumorigenic cell line, but was attained through progressive passages of BP-transformed cells. It was demonstrated that different patterns of changes in DNA content and chromatin organization may be involved in equally aggressive tumorigenic BP-transformed cell lines originated from the same cell line by different mechanisms.

Benzo(a)pyrene↗

PubMed1998

DNA content and chromatin texture of benzo[a]pyrene-transformed human breast epithelial cells as assessed by image analysis.

DNA content and chromatin supraorganization defined in terms of patterns of chromatin texture were studied by image analysis in Feulgen-stained human breast MCF-10F epithelial cells expressing different stages of tumorigenic progression after treatment with benzo[a]pyrene (BP). Nontransformed MCF-10F, nontumorigenic transformed BP1, and tumorigenic BP1-E1 cell lines were analyzed. A relationship between changes in DNA content and chromatin texture and the expression of different stages of tumorigenesis were researched in interphase nuclei. A significant decrease in Feulgen-DNA amounts and absorbing areas suggesting aneuploidy, and first detected in nontumorigenic transformed BP1 cells, became more relevant in the tumorigenic cell line BP1-E1. Simultaneously, an increase in optical densities and a change in related parameters, demonstrating increased chromatin higher order packing states, occurred in transformed BP1 cells and extended over larger chromatin areas in tumorigenic BP1-E1 cells. The results indicate loss of DNA and a change in chromatin higher order packing states accompanying the expression of different stages of the in vitro tumorigenesis process in BP-transformed human breast epithelial cells. It is suspected that the gradually acquired chromatin "condensation" supraorganization pattern is associated with activated transcription activities previously suggested for these cells.

Benzo(a)pyrene↗

PubMed1998

Nuclear and nucleolar image analysis of human breast epithelial cells transformed by benzo[a]pyrene and transfected with the c-Ha-ras oncogene.

Changes in nuclear and nucleolar morphometric parameters were investigated by image analysis procedures in human breast MCF-10F epithelial cells expressing different stages of the tumourigenic progression after benzo[a]pyrene (BP) transformation (BP1, BP1-E, and BP1-E1 cell lines), and additionally transfected with the c-Ha-ras oncogene (BP1-Tras cell line). Nuclear pleomorphism was evident in all the transformed cells. The analysis of different morphometric parameters did not show a clear relationship between specific nuclear and nucleolar changes and the expression of the different stages of the tumourigenesis, with the exception of the nucleolar size, which could be associated to the expression of the tumourigenic phenotype, and a nucleolar area/nuclear area ratio, which discriminated the immortalized, the transformed, and the tumourigenic phenotypes from one another. The nuclear morphometric data established for the BP-transformed cells and for the cells additionally transfected with the c-Ha-ras oncogene were suggestive of complex and distinct morphofunctional mechanisms involving the in vitro transformation of the MCF-10F cells. The nuclear changes found in the BP1-Tras cell line were assumed to be related to the additional effects and/or enhanced genomic instability induced by transfection with the ras oncogene.

Benzo(a)pyrene↗

PubMed1998

Peroxidase activity in Malpighian tubules of Triatoma infestans Klug.

Benzidine and diamino benzidine (DAB) oxidation, typically performed by peroxidases, was demonstrated by light and electron microscopy in peroxisomes, mitochondria and membranous structures which occurred in close contact with urate crystals in Malpighian tubules of nymphs and adults of Triatoma infestans. Peroxisomes were predominantly identified in cells of the distal region of the tubules, which is engaged in excretory mechanisms. DAB oxidation in mitochondria, even in the absence of hydrogen peroxide, may indicate the existence of a mitochondrial peroxidase and possibly a cytochrome c peroxidase. The localization of the extracellular membranous structures appeared restricted to the lumen of the proximal region of the tubules and they were assumed to be remnants of endoplasmic reticulum containing peroxidases.

Animals↗

PubMed1997

Acid phosphatase activity in Malpighian tubules of Triatoma infestans Klug.

Acid phosphatase activity was detected in the Malpighian tubules of the blood-sucking hemipteran, Triatoma infestans. The enzyme activity was especially prominent in the cytoplasmic globules which were assumed to be laminated 'concretions', which occur in the distal cells of the organ. It was also verified in the nuclei and in some cytoplasmic granules (lysosomes) of the proximal cells. The data indicated that lysosomes were involved with the nature or origin of the laminated concretions, but it is still questionable whether acid phosphatase activity exists in the nuclei.

Acid Phosphatase↗

PubMed1995

Modulation of ras transformation affecting chromatin supraorganization as assessed by image analysis.

Changes in chromatin supraorganization defined in terms of patterns of chromatin texture were studied by video image analysis in Feulgen-stained revertants of LTR-ras-transformed NIH 3T3 cells and in cell lines obtained by transfection of these revertants with sense and antisense constructs of the lysyl oxidase gene (also named Lox or "ras recision gene"). The objective was to determine whether changes in expression of the Lox gene, which have been assumed to modulate cell transformation by ras, could also affect the chromatin supraorganization changes known to be elicited in NIH 3T3 cells by ras transformation. The image analysis results revealed that, although a nuclear phenotype visually similar to the most frequent one (III) in ras-transformed NIH 3T3 cells also appeared in the revertant, it contained a remarkably less tight chromatin packing state. This situation was also found in the revertant transfected with the sense construct of the Lox gene, but in the revertant transfected with the Lox antisense constructs the chromatin texture of the III phenotype was equal to or close to that of the ras-transformed cells. With regard to the nuclear phenotype characterized by abundant loosely packed chromatin and less represented in the transformed cell lines (I'), changes in the various cell lines, although detectable, were not as drastic as those reported for the III phenotype. The enhancement in chromatin condensation of the type III nuclei, which affects euchromatin, is probably associated with a limited transcription of the genome. Although the image analysis results are mostly in agreement with previously published data on the molecular biology and tumorigenicity of the same cell lines, it appears that the phenomenon of chromatin condensation once established in NIH 3T3 cells by LTR-ras transformation could not be totally reverted by simply affecting Lox expression.

3T3 Cells↗

PubMed1995

Optical anisotropy of toluidine blue-stained transformed cell nuclei.

Changes in chromatin order with cell transformation were studied in terms of optical anisotropy (birefringence) in toluidine blue-stained NIH 3T3 and Balb/3T3 cells. The transformed NIH 3T3 cell lines used were obtained by transfection with the T24 cell H-ras oncogene and the whole genomic DNA of MCF-7 human breast carcinoma cells. C2PO and PAP2 cell lines were used as representatives of Ha-ras-transformed NIH 3T3 cells with poor and high metastatic ability, respectively. The chromatin of all cells examined exhibited metachromasy and a birefringence of greenish-yellow interference color. The highest birefringence intensity was found in the chromatin granules of the most frequent nuclear phenotype of ras- and MCF-7 DNA-transformed cells, with the exception of the cells with high metastatic ability, in which a faint birefringence was observed. The differences in chromatin birefringence intensity are assumed to indicate differences in chromatin stereoarrangement with cell transformation. In the case of the highly tumorigenic and/or highly metastatic transformants the faint birefringence is assumed to be associated with the heterogeneous and complex physiological processes that may require a relatively less ordered arrangement of the chromatin.

3T3 Cells↗

PubMed1995

Re-evaluating the AgNOR staining response in Triton X-100-treated liver cells by image analysis.

The response of liver cell nuclei to a variant of the AgNOR method, which includes a treatment with Triton X-100 prior to staining assumed to improve the quality of the AgNOR-positive images, was compared to that of the usual method by image analysis procedures. The objective was to determine whether Triton X-100 removes proteins from the nuclei to the point of affecting the AgNOR response. Statistically significant differences were not found in comparisons of a nuclear area/AgNOR-stained area ratio nor in the number of AgNOR-positive dots/aggregates. It was thus assumed that the Triton X-100 treatment did not remove nuclear proteins likely to affect the AgNOR response, though it induced a cleaner background and, consequently, a clearer contrast between the stained areas and the background.

Animals↗

PubMed1994

Critical electrolyte concentration and dispersion of birefringence in heterochromatin and euchromatin of copper sulfate-treated specimens of Triatoma infestans Klug.

Binding of toluidine blue molecules to DNA phosphates under Mg2+ competitive staining conditions and dispersion of birefringence were studied in heterochromatin and euchromatin of Malpighian tubule cells of the hemipteran, Triatoma infestans; 24 h after treatment with copper sulfate. The objective was to detect whether copper binds to chromatin DNA, affecting the critical electrolyte concentration (CEC) value and the chromatin anisotropy in these chromatin types. A decrease in CEC values as measured by metachromasy abolition, and in the concentration of Mg2+ leading to a change from anomalous dispersion of birefringence to normal dispersion of birefringence was found for the heterochromatin and euchromatin of two different nuclear types in the copper-treated material. Protein dissociation from DNA, leading to increased numbers of DNA phosphates available for dye binding, promoted by Mg2+ concentrations above that corresponding to the CEC point is suggested. Based on anisotropy findings, the binding of copper to DNA was assumed not to affect the usual molecular orientation of the DNA.

Animals↗

PubMed1994

Scanning microphotometry image analysis of Ha-ras-transformed human breast epithelial cells.

Nuclei of the human breast epithelial cells, MCF-10A, transfected with the c-Ha-ras oncogene, the ras proto-oncogene and the plasmid Homer 6 only, were studied by image analysis after Feulgen staining. This material had been previously used to demonstrate that the experimental insertion of the activated c-Ha-ras oncogene into the DNA of the MCF-10A cells induces their tumoural properties. The ras-transformed nuclei of the MCF-10A cells exhibited differences in chromatin supraorganization in comparison with the nuclei of human breast carcinoma MCF-7 cells, or c-Ha-ras-transformed NIH/3T3 cells and, to a much lesser extent, with those of other MCF-10A transfectants and the non-transfected MCF-10A cells. All MCF-10A transfectants exhibited unravelling of both condensed and non-condensed chromatin, which, however, was less drastic in the ras-transformed MCF-10A cells. It is hypothesized that simultaneous to a general chromatin loosening as a response to foreign transfected DNA, a reverse mechanism may be elicited by ras transformation in the chromatin of the MCF-10A cells. The result in terms of elicited chromatin condensation was not as strong as that promoted in ras-transformed NIH/3T3 cells. Considering that early steps of tumour progression in vitro have previously been assumed to be involved in the ras-transformed MCF-10A cells, the differences in chromatin supraorganization of the ras-transformed MCF-10A cells as compared with MCF-7 cells are probably due to their different tumoural stages plus the putative effect of the transfected DNA vector on the transfected MCF-10A cells.

Breast Neoplasms↗